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Na+-activated K+ channels express a large delayed outward current in neurons during normal physiology.

Budelli G, Hage TA, Wei A, Rojas P, Jong YJ, O'Malley K, Salkoff L - Nat. Neurosci. (2009)

Bottom Line: We found that TTX also eliminated this delayed outward component in rat neurons as a secondary consequence.Unexpectedly, we found that the activity of a persistent inward sodium current (persistent I(Na)) is highly effective at activating this large Na(+)-dependent (TTX sensitive) delayed outward current.These findings have far reaching implications for many aspects of cellular and systems neuroscience, as well as clinical neurology and pharmacology.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Washington University School of Medicine, St. Louis, Missouri, USA.

ABSTRACT
One of the largest components of the delayed outward current that is active under physiological conditions in many mammalian neurons, such as medium spiny neurons of the striatum and tufted-mitral cells of the olfactory bulb, has gone unnoticed and is the result of a Na(+)-activated K(+) current. Previous studies of K(+) currents in mammalian neurons may have overlooked this large outward component because the sodium channel blocker tetrodotoxin (TTX) is typically used in such studies. We found that TTX also eliminated this delayed outward component in rat neurons as a secondary consequence. Unexpectedly, we found that the activity of a persistent inward sodium current (persistent I(Na)) is highly effective at activating this large Na(+)-dependent (TTX sensitive) delayed outward current. Using siRNA techniques, we identified SLO2.2 channels as being carriers of this delayed outward current. These findings have far reaching implications for many aspects of cellular and systems neuroscience, as well as clinical neurology and pharmacology.

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Experiments in cells loaded with high [Na+]i5a: inhibition of delayed outward current by sodium efflux from an MSN. Effects of reversing the direction of TTX-sensitive sodium flux across the membrane. The intracellular pipette solution contains 40 mM Na+. 1. Control: Delayed outward currents plotted from the cell with normal [Na+]o. A series of control voltage-clamp step pulses were applied at 10 mV intervals to a maximum of +90 mV. 2. -[Na+]o: Outward currents plotted from the same cell after the removal of external Na+. This plot shows the delayed outward current in response to the same series of voltage-clamp step pulses after reversing the direction of Na+ current flow, which is now outward along the entire voltage range. 3. -[Na+]o + TTX: Outward currents plotted from the same cell after subsequently adding TTX to the 0[Na+]o condition in 2. above. This plot shows the delayed outward current in response to the same series of voltage-clamp step pulses after reducing the outward movement of Na+. Experiments shown in this figure were repeated seven times in MSNs and three times in tufted/mitral cells with similar results.5b: Conductance/voltage relations showing the normalized incremental conductance increases in the delayed outward current due to Na+ influx in cells loaded with different levels of [Na+]i. Tufted/mitral cells “loaded” with different intracellular concentrations of Na+, as indicated, were subjected to a series of voltage clamp step pulses. The incremental conductance/voltage curves plotted were constructed from the residual currents obtained by subtracting the currents recorded after removal of external Na+, from the control currents recorded in normal [Na+]o (as in Figure 5a, subtraction 1–2). As [Na+]i increases, the incremental conductance curves shift leftward to more negative voltages, and also show a steeper slope. This seems consistent with the hypothesis that, at higher concentrations of bulk [Na+]i, the sodium influx is augmenting the local Na+ concentration to yet a higher level. Conductances were calculated for individual cells at the three indicated concentrations, normalized to the maximum value, and then averaged. Regions of the curves to the left of Gmax were fit by a Boltzmann equation, shown in red. 0 mM [Na]int (■) V½ = 11.8 mV, n = 3; 20 mM [Na]int () V½ = 1.40 mV, ENa = 50.8 mV, n = 3. 30 mM [Na]int () V½ = 11.0 mV, ENa = 40.5 mV, n = 4.
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Figure 5: Experiments in cells loaded with high [Na+]i5a: inhibition of delayed outward current by sodium efflux from an MSN. Effects of reversing the direction of TTX-sensitive sodium flux across the membrane. The intracellular pipette solution contains 40 mM Na+. 1. Control: Delayed outward currents plotted from the cell with normal [Na+]o. A series of control voltage-clamp step pulses were applied at 10 mV intervals to a maximum of +90 mV. 2. -[Na+]o: Outward currents plotted from the same cell after the removal of external Na+. This plot shows the delayed outward current in response to the same series of voltage-clamp step pulses after reversing the direction of Na+ current flow, which is now outward along the entire voltage range. 3. -[Na+]o + TTX: Outward currents plotted from the same cell after subsequently adding TTX to the 0[Na+]o condition in 2. above. This plot shows the delayed outward current in response to the same series of voltage-clamp step pulses after reducing the outward movement of Na+. Experiments shown in this figure were repeated seven times in MSNs and three times in tufted/mitral cells with similar results.5b: Conductance/voltage relations showing the normalized incremental conductance increases in the delayed outward current due to Na+ influx in cells loaded with different levels of [Na+]i. Tufted/mitral cells “loaded” with different intracellular concentrations of Na+, as indicated, were subjected to a series of voltage clamp step pulses. The incremental conductance/voltage curves plotted were constructed from the residual currents obtained by subtracting the currents recorded after removal of external Na+, from the control currents recorded in normal [Na+]o (as in Figure 5a, subtraction 1–2). As [Na+]i increases, the incremental conductance curves shift leftward to more negative voltages, and also show a steeper slope. This seems consistent with the hypothesis that, at higher concentrations of bulk [Na+]i, the sodium influx is augmenting the local Na+ concentration to yet a higher level. Conductances were calculated for individual cells at the three indicated concentrations, normalized to the maximum value, and then averaged. Regions of the curves to the left of Gmax were fit by a Boltzmann equation, shown in red. 0 mM [Na]int (■) V½ = 11.8 mV, n = 3; 20 mM [Na]int () V½ = 1.40 mV, ENa = 50.8 mV, n = 3. 30 mM [Na]int () V½ = 11.0 mV, ENa = 40.5 mV, n = 4.

Mentions: We conducted experiments where the internal [Na+] was raised by filling whole cell patch recording electrodes with intracellular recording solutions containing 20, 30 and 40 mM Na+. In these experiments, we found that even though [Na+]i was elevated, additional Na+ influx during voltage clamp step pulses produced an incremental increase in the delayed outward current noted as a TTX-sensitive component. This was true even though sodium entry during voltage clamp step pulses was unlikely to appreciably raise the concentration of bulk [Na+]i. One possibility to explain the effectiveness of continued sodium entry during depolarizing step pulses is that persistent sodium entry in close proximity to Slack channels is the critical factor. We reasoned that if an inward Na+ current in close vicinity to the Slack channels can raise the local [Na+] to a higher level than the bulk intracellular solution, then an outward Na+ current in close proximity to the Slack channels may deplete the local [Na+]i. Thus, reversing the direction of TTX-sensitive sodium flux across the membrane in these Na+ loaded cells should result in a local [Na+]i which is lower than that achieved simply by blocking outward Na+ flux with TTX. Figure 5a shows the results of an experiment to test this possibility. To raise [Na+]i we filled the intracellular pipette with a solution containing 40 mM Na+, lowering K+ by an equal amount. A series of control voltage-clamp step pulses were then applied at 10 mV intervals to a maximum of +90 mV. We next reversed the direction of Na+ current flow along the entire voltage range by reducing the extracellular [Na+] to 0. After approximately a minute and a half, we repeated the series of voltage clamp step pulses and observed a substantially diminished delayed outward current. Finally, we added TTX to the extracellular solution containing 0 Na+ and, after approximately one minute and a half, we again repeated the series of voltage-clamp step pulses. At this final condition, the delayed outward current was larger, relative to the current with 0 mM [Na+]o. Our interpretation is that in the first series of control voltage clamp step pulses, Na+ entry raised the [Na+] in the vicinity of Slack channels to a level higher than that present in the bulk intracellular solution. However, after removing extracellular Na+, sodium moved in the outward direction across the membrane diluting the intracellular Na+ in the immediate vicinity of Slack channels to a lower level than would be present if the outward flow of Na+ was blocked. Finally, after adding TTX to the extracellular solution containing 0 Na+, the outward flow of Na+ was reduced and the concentration of Na+ in the vicinity of Slack channels rose to a level intermediate between control conditions, when the Na+ current was inward, and condition 2 when the Na+ current was outward. Figure 5a also shows the residual delayed outward currents obtained by subtracting the currents recorded after removal of external Na+, from the control currents recorded in normal [Na+]o, and the residual currents obtained by subtracting the currents recorded after addition of TTX to 0 mM external Na+, from the control currents recorded in normal [Na+]o (Fig. 5a). Notably, these residual currents decline at higher voltages. This is likely to be due to the fact that Slack channels at higher voltages are particularly vulnerable to block by [Na+]i (6,11). This could also be due to the fact that the sodium current was outward at the higher voltage steps for the control currents recorded in normal [Na+]o and therefore the outward flow of Na+ could partially deplete the local concentration of Na+ sensed by SLO2 channels. However, we have noted that the reduction of outward current after TTX is added (as in Fig. 1a–c) is not immediate and requires approximately 1 to 2 min. before the current is stabilized at the lower level. Thus, the local concentration of Na+ sensed by SLO2 channels may only be partially changed during shorter voltage clamp step pulses.


Na+-activated K+ channels express a large delayed outward current in neurons during normal physiology.

Budelli G, Hage TA, Wei A, Rojas P, Jong YJ, O'Malley K, Salkoff L - Nat. Neurosci. (2009)

Experiments in cells loaded with high [Na+]i5a: inhibition of delayed outward current by sodium efflux from an MSN. Effects of reversing the direction of TTX-sensitive sodium flux across the membrane. The intracellular pipette solution contains 40 mM Na+. 1. Control: Delayed outward currents plotted from the cell with normal [Na+]o. A series of control voltage-clamp step pulses were applied at 10 mV intervals to a maximum of +90 mV. 2. -[Na+]o: Outward currents plotted from the same cell after the removal of external Na+. This plot shows the delayed outward current in response to the same series of voltage-clamp step pulses after reversing the direction of Na+ current flow, which is now outward along the entire voltage range. 3. -[Na+]o + TTX: Outward currents plotted from the same cell after subsequently adding TTX to the 0[Na+]o condition in 2. above. This plot shows the delayed outward current in response to the same series of voltage-clamp step pulses after reducing the outward movement of Na+. Experiments shown in this figure were repeated seven times in MSNs and three times in tufted/mitral cells with similar results.5b: Conductance/voltage relations showing the normalized incremental conductance increases in the delayed outward current due to Na+ influx in cells loaded with different levels of [Na+]i. Tufted/mitral cells “loaded” with different intracellular concentrations of Na+, as indicated, were subjected to a series of voltage clamp step pulses. The incremental conductance/voltage curves plotted were constructed from the residual currents obtained by subtracting the currents recorded after removal of external Na+, from the control currents recorded in normal [Na+]o (as in Figure 5a, subtraction 1–2). As [Na+]i increases, the incremental conductance curves shift leftward to more negative voltages, and also show a steeper slope. This seems consistent with the hypothesis that, at higher concentrations of bulk [Na+]i, the sodium influx is augmenting the local Na+ concentration to yet a higher level. Conductances were calculated for individual cells at the three indicated concentrations, normalized to the maximum value, and then averaged. Regions of the curves to the left of Gmax were fit by a Boltzmann equation, shown in red. 0 mM [Na]int (■) V½ = 11.8 mV, n = 3; 20 mM [Na]int () V½ = 1.40 mV, ENa = 50.8 mV, n = 3. 30 mM [Na]int () V½ = 11.0 mV, ENa = 40.5 mV, n = 4.
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Figure 5: Experiments in cells loaded with high [Na+]i5a: inhibition of delayed outward current by sodium efflux from an MSN. Effects of reversing the direction of TTX-sensitive sodium flux across the membrane. The intracellular pipette solution contains 40 mM Na+. 1. Control: Delayed outward currents plotted from the cell with normal [Na+]o. A series of control voltage-clamp step pulses were applied at 10 mV intervals to a maximum of +90 mV. 2. -[Na+]o: Outward currents plotted from the same cell after the removal of external Na+. This plot shows the delayed outward current in response to the same series of voltage-clamp step pulses after reversing the direction of Na+ current flow, which is now outward along the entire voltage range. 3. -[Na+]o + TTX: Outward currents plotted from the same cell after subsequently adding TTX to the 0[Na+]o condition in 2. above. This plot shows the delayed outward current in response to the same series of voltage-clamp step pulses after reducing the outward movement of Na+. Experiments shown in this figure were repeated seven times in MSNs and three times in tufted/mitral cells with similar results.5b: Conductance/voltage relations showing the normalized incremental conductance increases in the delayed outward current due to Na+ influx in cells loaded with different levels of [Na+]i. Tufted/mitral cells “loaded” with different intracellular concentrations of Na+, as indicated, were subjected to a series of voltage clamp step pulses. The incremental conductance/voltage curves plotted were constructed from the residual currents obtained by subtracting the currents recorded after removal of external Na+, from the control currents recorded in normal [Na+]o (as in Figure 5a, subtraction 1–2). As [Na+]i increases, the incremental conductance curves shift leftward to more negative voltages, and also show a steeper slope. This seems consistent with the hypothesis that, at higher concentrations of bulk [Na+]i, the sodium influx is augmenting the local Na+ concentration to yet a higher level. Conductances were calculated for individual cells at the three indicated concentrations, normalized to the maximum value, and then averaged. Regions of the curves to the left of Gmax were fit by a Boltzmann equation, shown in red. 0 mM [Na]int (■) V½ = 11.8 mV, n = 3; 20 mM [Na]int () V½ = 1.40 mV, ENa = 50.8 mV, n = 3. 30 mM [Na]int () V½ = 11.0 mV, ENa = 40.5 mV, n = 4.
Mentions: We conducted experiments where the internal [Na+] was raised by filling whole cell patch recording electrodes with intracellular recording solutions containing 20, 30 and 40 mM Na+. In these experiments, we found that even though [Na+]i was elevated, additional Na+ influx during voltage clamp step pulses produced an incremental increase in the delayed outward current noted as a TTX-sensitive component. This was true even though sodium entry during voltage clamp step pulses was unlikely to appreciably raise the concentration of bulk [Na+]i. One possibility to explain the effectiveness of continued sodium entry during depolarizing step pulses is that persistent sodium entry in close proximity to Slack channels is the critical factor. We reasoned that if an inward Na+ current in close vicinity to the Slack channels can raise the local [Na+] to a higher level than the bulk intracellular solution, then an outward Na+ current in close proximity to the Slack channels may deplete the local [Na+]i. Thus, reversing the direction of TTX-sensitive sodium flux across the membrane in these Na+ loaded cells should result in a local [Na+]i which is lower than that achieved simply by blocking outward Na+ flux with TTX. Figure 5a shows the results of an experiment to test this possibility. To raise [Na+]i we filled the intracellular pipette with a solution containing 40 mM Na+, lowering K+ by an equal amount. A series of control voltage-clamp step pulses were then applied at 10 mV intervals to a maximum of +90 mV. We next reversed the direction of Na+ current flow along the entire voltage range by reducing the extracellular [Na+] to 0. After approximately a minute and a half, we repeated the series of voltage clamp step pulses and observed a substantially diminished delayed outward current. Finally, we added TTX to the extracellular solution containing 0 Na+ and, after approximately one minute and a half, we again repeated the series of voltage-clamp step pulses. At this final condition, the delayed outward current was larger, relative to the current with 0 mM [Na+]o. Our interpretation is that in the first series of control voltage clamp step pulses, Na+ entry raised the [Na+] in the vicinity of Slack channels to a level higher than that present in the bulk intracellular solution. However, after removing extracellular Na+, sodium moved in the outward direction across the membrane diluting the intracellular Na+ in the immediate vicinity of Slack channels to a lower level than would be present if the outward flow of Na+ was blocked. Finally, after adding TTX to the extracellular solution containing 0 Na+, the outward flow of Na+ was reduced and the concentration of Na+ in the vicinity of Slack channels rose to a level intermediate between control conditions, when the Na+ current was inward, and condition 2 when the Na+ current was outward. Figure 5a also shows the residual delayed outward currents obtained by subtracting the currents recorded after removal of external Na+, from the control currents recorded in normal [Na+]o, and the residual currents obtained by subtracting the currents recorded after addition of TTX to 0 mM external Na+, from the control currents recorded in normal [Na+]o (Fig. 5a). Notably, these residual currents decline at higher voltages. This is likely to be due to the fact that Slack channels at higher voltages are particularly vulnerable to block by [Na+]i (6,11). This could also be due to the fact that the sodium current was outward at the higher voltage steps for the control currents recorded in normal [Na+]o and therefore the outward flow of Na+ could partially deplete the local concentration of Na+ sensed by SLO2 channels. However, we have noted that the reduction of outward current after TTX is added (as in Fig. 1a–c) is not immediate and requires approximately 1 to 2 min. before the current is stabilized at the lower level. Thus, the local concentration of Na+ sensed by SLO2 channels may only be partially changed during shorter voltage clamp step pulses.

Bottom Line: We found that TTX also eliminated this delayed outward component in rat neurons as a secondary consequence.Unexpectedly, we found that the activity of a persistent inward sodium current (persistent I(Na)) is highly effective at activating this large Na(+)-dependent (TTX sensitive) delayed outward current.These findings have far reaching implications for many aspects of cellular and systems neuroscience, as well as clinical neurology and pharmacology.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Washington University School of Medicine, St. Louis, Missouri, USA.

ABSTRACT
One of the largest components of the delayed outward current that is active under physiological conditions in many mammalian neurons, such as medium spiny neurons of the striatum and tufted-mitral cells of the olfactory bulb, has gone unnoticed and is the result of a Na(+)-activated K(+) current. Previous studies of K(+) currents in mammalian neurons may have overlooked this large outward component because the sodium channel blocker tetrodotoxin (TTX) is typically used in such studies. We found that TTX also eliminated this delayed outward component in rat neurons as a secondary consequence. Unexpectedly, we found that the activity of a persistent inward sodium current (persistent I(Na)) is highly effective at activating this large Na(+)-dependent (TTX sensitive) delayed outward current. Using siRNA techniques, we identified SLO2.2 channels as being carriers of this delayed outward current. These findings have far reaching implications for many aspects of cellular and systems neuroscience, as well as clinical neurology and pharmacology.

Show MeSH